The structure and function of bacterial protein toxins are analyzed using biochemical, genetic, and cell biology methods. Our previous work showed that the anthrax toxin protective antigen protein (PA, 83 kDa) binds to recep-tors on the surface of sensitive cells, is cleaved by a cell surface protease, probably furin, and only then captures either of the two other proteins, lethal factor (LF, 90 kDa) or edema factor (EF, 89 kDa). The PA-LF and PA-EF complexes enter cells by endocyto-sis and reach an acidic compartment from which LF and EF escape to the cytosol. EF is a calcium- and calmodulin-depen-dent adenylyl cyclase which causes large and unregulated increases in intra-cellular cAMP concentra-tions. An important advance was made this year through a collaboration with George Vande Woude, NCI. It was found that LF is a metalloprotease that rapidly cleaves mitogen-activated protein kinase kinase 1 (MAPKK1, or MEK1). Cleavage occurs near the N-terminus, after residue 7, and inactivates MEK1. It will now be possible to screen for protease inhibitors that block the action of LF and limit the pathogenesis of anthrax infection.The collaborative study which determined the structure of PA was extended to determine the structure of LF. Large amounts of LF were prepared from a fusion protein in which residues 1-164 of PA are attached to the N-terminus of LF. Analysis was completed of a large set of mutated PA proteins in which two flexible loops in the receptor-binding domain (domain IV) were randomly substituted by codon mutagenesis with Ala. Two mutants unable to bind to cells identified a region on the surface of PA that appears to interact with receptor.Chemical and retroviral mutagenesis of CHO cells yielded mutants resistant to Clostridium septicum alpha toxin, a hemolysin related to the better-known aerolysin. Analysis showed that the alpha toxin resistant CHO mutants had lost the ability to synthesize glycosylphosphatidylinositol (GPI)-anchors, and the mutation was localized to the second enzyme in the GPI biosynthetic pathway. Comparison of a number of cell lines confirmed that alpha toxin uses (GPI)-anchored proteins as receptors, but the set of proteins is not exactly the same set as used by aerolysin.